Breast cancer is the third most common cancer world wide and the most common cancer of women, affecting 1 in 8 in the western world (1,2). In the USA, breast cancer is the second leading cause of female cancer mortality accounting for about 10% of all cancer deaths (3). In breast cancer, as in other cancers, metastasis is the main cause of death in most patients. To date, several breast cancer metastasis-associated genes have been identified (for review, see reference 2). However, indirect measures of metastatic progression, including assessment of intratumoral vascular invasion, presence or absence of lymph node involvement and size of the primary carcinoma remain the most widely used methods for the assessment of breast cancer progression. Electro-diagnosis has also been practised, although its cellular/molecular basis remains unknown (4).
We have shown previously that the functional expression of a voltage-gated Na+ channel (VGSC) can distinguish strongly and weakly metastatic human and rat prostatic cancer cells (5,6) and that VGSC activity contributes to cellular behaviours integral to metastasis, including cellular process extension (7), lateral motility (8), transverse invasion (5,6,9) and secretory membrane activity (10). Carcinomas of the breast and prostate have some similar features, including hormone-sensitivity, a pronounced tropism for metastasis to bone and tendency for co-occurrence in families (11).
Voltage-gated Na+ channels (VGSCs)1 are composed of a large (≅240 kD), four-transmembrane domain α-subunit (VGSCα) and several different auxiliary β-subunits (VGSCβs) (Catterall, W. A. (1986) Ann. Rev. Biochem. 55, 953-985). Expression of the VGSCα alone is sufficient for functional channel formation (Goldin, A. L et al (1986). Proc. Natl. Acad. Sci USA 83, 7503-7507). The VGSCβ(s) serve a number of supporting roles such as facilitating functional channel availability (Isom, L. L., et al (1995) Cell 83, 433-442), modulating channel kinetics (Isom, L. L., et al (1992) Science 256, 839-842, Cannon, S. C., et al (1993) Pflugers Arch. 423, 155-157) and perhaps even altering pharmacological characteristics (Bonhaus, D. W., et al (1996) Neuropharmacol. 35, 605-613) (see also (15)).
VGSCαs constitute a family of at least twelve different genes in higher vertebrates (Plummer, N. W. and Meisler, M. H. (1998) Genomics 57, 323-331; 17), denoted SCN1A to SCN11A; their products have been cloned from a variety of excitable cell types. Their specific expressions are under dynamic, spatio-temporal control. At least two subfamilies of VGSCα genes have been described based on sequence data: Nav1 and Nav2 (George, A. L., et al (1992) Proc. Natl. Acad. Sci. USA 89, 4893-4897). Although not yet experimentally determined, it is generally held that these subfamilies represent VGSCαs with markedly different electro-physiological properties (Akopian, A. N., et al (1997) FEBS Letts. 400, 183-187). In fact the lack of conservation of landmark VGSCα sequences in Nav2 VGSCαs implies that they may not even be voltage-gated or Na+ selective (Akopian, A. N., et al (1997) FEBS Letts. 400, 183-187, Schlief, T., et al (1996) Eur. Biophys. J. 25, 75-91). The existence of a third subfamily, Nav3, has recently been proposed with the cloning of a cDNA (NaN/SNS2) from rat dorsal root ganglion (DRG) cells. Although NaN/SNS2 shares less than 50% sequence homology with other VGSCαs, its deduced amino acid sequence possesses all the characteristic sequences of Nav1 VGSCαs (Dib-Hajj, S. D, et al (1998) Proc. Natl. Acad. Sci. USA 95, 8963-8968).
By utilizing RT-PCR and in situ hybridization methods, several studies have documented the simultaneous expression of multiple VGSCαs within diverse cell types (Black, J. A., et al (1994) Mol. Brain Res. 23, 235-245; Dib-Hajj, S. D., et al (1996) FEBS Letts. 384, 78-82; Fjell, J., et al (1999) Mol. Brain Res. 67, 267-282). Particular VGSCαs have been found to be expressed at different levels, with expression under dynamic control (e.g. during development or injury). For example, mRNAs for at least eight different VGSCαs were found in adult rat DRG cells, with a wide range of expression levels: RB1, Na6, NaN/SNS2 and SCL-11 mRNAs were expressed at very high levels, PN1 and SNS/PN3 at intermediate levels, and RB2 and RB3 at very low levels (Dib-Hajj, S. D, et al (1998) Proc. Natl. Acad. Sci. USA 95, 8963-8968; Black, J. A., et al (1996) Molec. Brain Res. 43, 117-131; Sangameswaren, L., et al (1997) J. Biol. Chem. 272, 14805-14809). Following axonal injury SNS and NaN/SNS2 mRNAs were dramatically down-regulated, whilst expression of RB1, RB2 and RB3 was up-regulated (Dib-Hajj, S., et al (1996) Proc. Natl. Acad. Sci. USA 93, 14950-14954; Dib-Hajj, S. D., et al (1998) J. Neurophysiology 79, 2668-2676).
VGSCα genes can occur as a number of alternatively spliced isoforms, expression of which is also under dynamic control. Alternative splicing of exons coding for the third segment (S3) of the first transmembrane domain (D1) has been found to be developmentally regulated for SCN2A and SCN3A (19, 20), yielding “neonatal” and “adult” forms. These code for proteins which differ by only one amino acid, positioned at the extreme extracellular end of S3. The effect of this change on VGSCα function is presently unclear. Similar alternatively spliced exons exist at the corresponding position in SCN8A and SCN9A (Belcher, S. M et al (1995) Proc. Natl. Acad. Sci. USA 92, 11034-11038; Plummer, N. W., et al (1998) Genomics 54, 287-296) but not in SCN4A, SCN5A, SCN10A and SCN11A (George, A. L., et al (1993) Genomics 15, 598-606; Wang, D. W., et al (1996) Biophys. J. 70, 238-245; Souslova, V. A., et al (1997) Genomics 41, 201-209; Dib-Hajj, S. D., et al (1999) Genomics 59, 309-318). To date, no evidence of such alternative splicing has been found for SCN1A or SCN7A. Alternative splicing also occurs in other regions of the VGSCα, particularly inter-domain (ID) 1-2 and D3.
The strict regulation of multiple VGSCα gene and splice product expression within the available VGSCα mRNA pool, among different tissue types and during development or following injury (e.g. Dib-Hajj, S., et al (1996) Proc. Natl. Acad. Sci. USA 93, 14950-14954; Dib-Hajj, S. D., et al (1998) J. Neurophysiology 79, 2668-2676; Kallen, R. G., et al (1990) Neuron 4, 233-242) would suggest that different VGSCα gene products and their isoforms are likely to have significantly different functional roles, which, at present, are largely unknown.
The functional roles of VGSCs are best understood in the central nervous system where VGSC activity controls not only basic impulse generation and conduction but also directional and patterned growth, including target-specific axonal migration and regional synaptic connectivity (Catalano, S. M. and Shatz, C. J. (1998) Science 281, 559-562; Penn, A. A., et al (1998) Science 279, 2108-2112; Shatz, C. J. (1990) Neuron 5, 745-756). VGSCs have also been implicated in several hereditary diseases of excitable tissues (Plummer, N. W. and Meisler, M. H. (1998) Genomics 57, 323-331; Zhou, J. and Hoffman, E. P. (1994) J. Biol. Chem. 269, 18563-18571), and in more complicated pathological disorders, including chronic pain syndromes (Tanaka, M., et al (1998) NeuroReport 9, 967-972), epilepsy (Bartolomei, F., et al (1997) J. Neurocytol. 26, 667-678), ischaemic stroke (Skaper, S. D., et al (1998) FASEB J. 12, 725-731) and Alzheimer's disease (Kanazirska, M., et al (1997) Biochem. Biophys. Res. Comm. 232, 84-87). There is increasing evidence that VGSC expression is also associated with strong metastatic potential in rat (MAT-LyLu) and human (PC-3) models of prostate cancer (Grimes, J. A., et al (1995) FEBS Letts. 369, 290-294; Laniado, M., et al (1997) Am. J. Pathol. 150, 1213-1221; Smith, P., et al (1998) FEBS Letts 423, 19-24). Indeed, expression of functional VGSCs may have a direct, positive influence upon the metastatic process. Accordingly, blockage of VGS currents in these strongly metastatic cell lines, by application of tetrodotoxin (TTX), significantly (˜30%) reduced the cells' invasive potential. Electro-physiological and pharmacological properties of the current in the rat were consistent with the channels being neuronal, TTX-sensitive (Nav1) type (Grimes, J. A. and Djamgoz, M. B. A. (1998) J. Cell. Physiol. 175, 50-58). SCN4A gene expression was found in both strongly and weakly metastatic cell lines of human and rat (Diss, J. K. J., et al (1998) FEBS Letts 427, 5-10). However, the pharmacological properties of the VGS currents in the rat MAT-LyLu cells were not consistent with those reported for this VGSCα. This could result from (1) the numerous differences determined in the MAT-LyLu/AT-2 rSkMl primary sequence; (2) differences in post-translational mechanisms (eg association with auxiliary subunits, level of glycosylation/phosphorylation of the channel) in these cells; or (3) the presence of other VGSCαs in the MAT-LyLu cells that produce the recorded VGS currents.
The present study aimed to determine (i) whether mRNA and functional protein expression of VGSCs differed between strongly and weakly metastatic breast cancer cells; (ii) which member(s) of the VGSCα family was responsible for the voltage-gated Na+ (VGS) currents detected; and (iii) whether the VGSCα expression pattern found in in vitro models would also be reflected in human breast cancer biopsy tissues. These aspects were studied using electrophysiological and reverse-transcription polymerase chain reaction (RT-PCR) based techniques. Initially, two robust breast cancer cell lines of contrasting metastatic aggressiveness were adopted: the strongly metastatic MDA-MB-231 cells and the weakly metastatic MCF-7 cells (18,19). The mRNA(s) responsible for the functional VGSC α-subunit expression was determined. Finally, VGSC mRNA expression was also investigated in frozen biopsy tissues of different clinical grade to test whether VGSCα occurrence could also be correlated with cancer progression in vivo.
Materials and Methods
Cell culture. MDA-MB-231 and MCF-7 cells were grown and maintained in Dulbecco's modified Eagle's medium (Life Technologies Ltd, Paisley, UK) supplemented with 4 mM L-glutamine and 10% foetal bovine serum. Cells were seeded into 100 mm Falcon tissue culture dishes (Becton Dickinson Ltd, Plymouth, UK) and grown in an incubator at 37° C., 100% humidity and 5% CO2.
Electrophysiology. Patch pipettes (of normal resistances between 5-15 MΩ) were filled with a solution containing (in mM) NaCl 5, KCl 145, MgCl2 2, CaCl2 1, HEPES 10 and EGTA 11, adjusted to pH 7.4 with 1 M KOH. Whole-cell membrane currents were recorded from cells that appeared ‘isolated’ in culture using an Axopatch 200B (Axon Instruments) amplifier. Analogue signals were filtered at 5 KHz using a low-pass Bessel filter. Signals were sampled at 5 KHz and digitised using a Digidata (1200) interface. Data acquisition and analysis of whole-cell currents were performed using pClamp (Axon Instruments) software. Holding potentials of −90 mV or −100 mV were used to study K+ and Na+ currents, respectively, unless stated otherwise. Resting potentials were measured immediately following attainment of the ‘whole-cell’ recording configuration. Experiments on both MDA-MB-231 and MCF-7 cells were performed on three separate dishes which had been plated for between 1-3 days.
Two basic command voltage protocols were used to study the electrophysiological and pharmacological properties of the Na+ and K+ currents, as follows:                1. Current-voltage (1-V) protocol. This protocol was used to study the voltage-dependence of Na+ and K+ channel activation. Cells were pulsed to depolarising test potentials between −70 and +60 mV, in 5 mV steps. The test pulse duration was 40 ms (Na+ currents) or 200 ms (K+ currents); the interpulse period was 20 s.        2. Repeat single-pulse protocol. This was used to monitor the effects of drugs on current amplitude. Test pulses were to −10 mV (Na+ currents) or +60 mV (K+ currents). The test pulse duration was 40 ms (Na+ currents) or 200 ms (K+ currents); the interpulse duration was 20 s and there were 5 repeat pulses.        
Pharmacology. Tetrodotoxin (TTX), purchased from Alomone Labs Ltd (Jerusalem, Israel), was made as a stock solution (×1000) in the external bath solution, frozen at −20° C., defrosted and diluted as required. Briefly, TTX was back-loaded into a glass capillary (with a tip size of ˜5 μm). The glass capillary was then connected to a pneumatic picopump (PV 800, WP Instruments), mounted on a microdrive (Lang-Electronik, Huttenberg, Germany) and manoeuvred to within ˜10 μm of the cell under investigation.
The effect of TTX on the inward current (1) has been presented as the percentage block of current (B) in comparison to the control values, calculated as follows:B(%)=[(Iafter−Ibefore)/Ibefore]×100
VGSCα degenerate primer screens. Total cellular RNA was isolated from two batches of each of the cell lines by the acid guanidium thiocyanate-phenol-chloroform method (20) or as described below. Briefly, cells were homogenized in a solution (“A”), using an IKA homogeniser, such that 1 ml of solution was used per 0.1 g of tissue. Solution A contained 4 M guanidinium thiocyanate, 25 mM Na+ citrate (pH 7.0), 0.5% sarcosyl and 0.72% (v/v) β-mercaptoethanol. The following were then added and shaken vigorously for 10 seconds: 2 M Na+ acetate, pH 4.0 (10% volume of solution A), phenol (equal volume of solution A) and chloroform (20% volume of solution A). Centrifugation was performed at 10,000×g for 20 mins at 4° C. The supernatant was taken and precipitated with isopropanol. Then, the samples were centrifuged as before and the pellet was resuspended in about 30% of the initial volume of solution A. A second isopropanol precipitate was performed, the pellet was washed with 75% ethanol, and resuspended in sterile distilled water.
Screens were then performed on each of the four extracts, as described previously (21 and GB 0021617.6, supra), using VGSCα degenerate PCR primers, YJ1 and YJ2C (Table 1A).
Twenty five clones with “inserts” were selected by gel electrophoresis for each of the RNA extracts. A subset of the twenty five clones with inserts, derived from each cell line, were then sequenced using the Amersham Thermo Sequenase fluorescent cycle sequencing kit and the Vistra DNA 725 automated sequencer. Sequences were identified by searching the GenBank DNA database using BLAST 2.0.8 (22). Oligonucleotide primers specific for scn5a and scn9a VGSCαs, identified by the sequencing, were subsequently designed (Table 1A). These worked in conjunction with the Universal vector primers and permitted rapid PCR screening of all other clones without the need for sequencing. PCRs using these primers were initially tested on sequenced clones to confirm that they yielded only specific products. Rapid screening PCR reactions were then performed as in (21) and GB 0021617.6, supra. Products were analysed by gel electrophoresis on 0.8% agarose gels. Minipreps that did not test positive for these VGSCαtypes were sequenced to determine identity.
TABLE 1PCR primers used in (A) degenerate VGSCα primer screening,(B) specific PCRs and (C) SQT-PCRs (numbering according to GenBank).Primer annealing positions are indicated in parentheses.A. Degenerate VGSCα Primer ScreeningYJ1: -5′ GCGAAGCTT(C/T)TGG(C/T)TIATITT(C/T)I(A/C/G/T)IAT(A/T/C)ATGGG 3′(SEQ ID NO 7) YJ2C: -5′ ATAGGATCCAICCI(A/C/G/T)I(A/G)AAIGC(A/C/G/T)AC(C/T)TG 3′(40° C.)(SEQ ID NO 8) Scn5a-P1: -5′ TACAATTCTCCGGTCAAGTT 3′ (4312-4331; 56° C.)(SEQ ID NO 9) Scn9a-P1: -5′ ATGTTAGTCAAAATGTGCGA 3′ (4139-4158; 54° C.)(SEQ ID NO 10) B. Specific PCR TestsScn5a-P2: -5′ CATCCTCACCAACTGCGTGT 3′ (570-589)(SEQ ID NO 11) Scn5a-P3: -5′ CACTGAGGTAAAGGTCCAGG 3′ (1059-1078; 58° C.)(SEQ ID NO 12) Scn8a-P1: -5′ AGACCATCCGCACCATCCTG 3′ (3855-3874)(SEQ ID NO 13) Scn8a-P2: -5′ TGTCAAAGTTGATCTTCACG 3′ (4351-4370; 60° C.)(SEQ ID NO 14) Scn9a-P2: -5′ TATGACCATGAATAACCCGC 3′ (474-493)(SEQ ID NO 15) Scn9a-P3: -5′ TCAGGTTTCCCATGAACAGC 3′ (843-862; 59° C.)(SEQ ID NO 16) hCytb5R-P1: -5′ TATACACCCATCTCCAGCGA 3′ (299-318)(SEQ ID NO 17) hCytb5R-P2: -5′ CATCTCCTCATTCACGAAGC 3′ (771-790; 60° C.)(SEQ ID NO 18) C. SQT-PCRsScn5a-P4: -5′ CTGCTGGTCTTCTTGCTTGT 3′ (2896-2915)(SEQ ID NO 19) Scn5a-P5: -5′ GCTGTTCTCCTCATCCTCTT 3′ (3329-3348; 60° C.)(SEQ ID NO 20) Scn8a-P3: -5′ AACCCTATTCCGAGTCATCC 3′ (3827-3846)(SEQ ID NO 21) Scn8a-P4: -5′ TGCACTTTCCTCTGTGGCTA 3′ (4325-4344; 60° C.)(SEQ ID NO 22) Scn9a-P4: -5′ AAGGAAGACAAAGGGAAAGA 3′ (5941-5960)(SEQ ID NO 23) Scn9a-P5: -5′ TCCTGTGAAAAGATGACAAG 3′ (6289-6308; 56° C.)(SEQ ID NO 24)
VGSCα-specific PCR tests. These were performed, as in (21) and GB 0021617.6 in order to ensure that the VGSCαs found in the degenerate primer screens were truly expressed in the respective cell lines (and notproduced from contaminating genomic DNA).
Briefly, DNA was removed from the extracts by digestion with DNase 1 and 5 μg of the total RNA was used as the template for single-stranded cDNA (sscDNA) synthesis (Superscript II, GIBCO BRL). sscDNA synthesis was primed with a random hexamer mix (R6) in a final volume of 20 μl. VGSCα cDNA was then amplified from the R6-sscDNA pool by PCR (Taq DNA polymerase, Amersham Pharmacia) using degenerate PCR primers (YJ1 and YJ2C) used previously to amplify both Nav1 and Nav2 VGSCαs from adult rat retinal pigment epithelial cells (Dawes, H., et al (1995) Vis. Neurosci. 12, 1001-1005), and novel VGSCαs from a protochordate ascidian (Okamura, Y., et al (1994) Neuron 13, 937-948). PCR reactions were performed on 4 μl of the R6-sscDNA template, using 200 μM of each dNTP, 1 unit of Taq, 1×Taq buffer and 1 μM of each primer, in a final volume of 20 μl. Amplification was via: (i) initial denaturation at 94° C. for 5 min; (ii) addition of 1 U enzyme; (iii) 33-35 cycles of denaturation at 94° C. for 1 min, annealing at 40° C. for 1 min, and elongation at 72° C. for 1 min; and (iv) elongation at 72° C. for 10 min. For this and all PCRs performed, reactions with no sscDNA added were also carried out to control for cross-contamination from other DNA sources.
PCR products were analysed by electrophoresis and gel purified prior to ligation into the pGEM-T vector (pGEM-T Easy Vector System, Promega). These were then used to transform E. coli (pMosBlue, Amersham). Plasmid DNA was recovered from bacterial cultures using a modified version of the Vistra Labstation 625 miniprep procedure (Vistra DNA Systems, Amersham).
Reactions designed to amplify specific VGSCαs were performed on both strongly and weakly metastatic cell line extracts, irrespective of whether these subunits had previously been found in degenerate screens. The primer sequences and reaction annealing temperatures used are shown in Table 1B. Evident products were cloned and sequenced, and a consensus sequence for each VGSCα in each cell line then produced (using at least three clones).
Semi-quantitative PCR (SQT-PCR). SQT-PCRs based on kinetic observation of reactions were performed similarly to (21) and GB 0021617.6.
DNased RNA extracts were used to produce sets of R6-sscDNAs for each extract. 2.4 μl of these k6-sscDNAs was used as the template for VGSCα-specific PCRs (performed as above), in a final volume of 60 μl. To allow direct comparison of results obtained from strongly and weakly metastatic cell lines, all comparable R6-sscDNA and PCR reactions were performed simultaneously. ‘Blanks’, with no template added, were used as controls. PCRs were performed using different 20-mer primers for each of the three VGSCαs which did not amplify multiple VGSCα products derived from different splice variants (unlike the specific PCRs above). The primers and annealing temperatures of the PCRs used are shown in Table 1C. scn8a and scn9a VGSCα products did not span conserved intron sites so control PCR reactions were performed for these SQT-PCRs in which the sscDNA template was replaced by an aliquot from a reverse transcription reaction which had no reverse transcriptase added. All products were cloned and sequenced, as above, to ensure that only VGSCα-specific products were amplified.
A kinetic observation approach (45; Hoof et al (1991) Anal. Biochem. 196, 161-169; Wiesner et al (1992) Biochem. Biophys. Res. Comm. 183, 553-559) was adopted such that an aliquot of 5 μl from the 60 μl reaction was taken at the end of each amplification cycle, for eleven cycles, while reactions were held at 72° C. The amplification cycle at which aliquots were first taken differed depending on the VGSCα studied. These aliquots were then electrophoresed (0.8% agarose gels) with DNA markers of known concentration. Gels were post-stained for 15 minutes (TBE buffer containing 0.8 μg/ml ethidium bromide), and digitally imaged (GDS 7500 Advanced Gel Documentation System, Ultra-Violet Products). Total product mass (nanograms) in each aliquot was determined by image analysis (1D Image Analysis Software, Kodak Digital Science). Two characteristic stages in each PCR reaction were quantified:                (1) Threshold PCR cycle number (CNt) at which a given PCR product could just be detected by the image analysis software (default settings).        (2) PCR cycle number at which the exponential phase of the reaction finished (CNe).        
Accumulation of reaction product with increasing PCR cycle number follows a sigmoid curve (Kohler, T. (1995). Quantitation of mRNA by Polymerase Chain Reaction, pp 3-14, eds. Kohler, T., Lassner, D., Rost, A.-K., Thamm, B., Pustowoit, B. and Remke, H. (Springer, Heidelberg)). However, the two extremes of this curve were unknown or undetermined for the SQT-PCR data (i.e. the initial mass of cDNA at zero cycles was unknown, and the final product mass at the end of the PCR undetermined). Thus, a sigmoid curve could not be fitted to the data. Instead a third-order polynomial equation, which also has only one possible point of inflexion (here corresponding to the end of the exponential phase of the PCR), was used to approximate a sigmoid curve. Curve-fitting was performed using STATISTICA (SoftStat Inc.), and the second derivative then calculated, to give CNe. This procedure could be performed successfully, with the calculated values of CNe falling within the data points obtained experimentally (FIG. 1). Data are presented as means and standard errors for each cell line (three repeats on two extracts for each VGSCα). The values of CNt and CNe were used directly to compare the levels of expression of each VGSCα in the strongly and weakly metastatic cell lines.
Mean CNt values were calculated for each of the VGSCαs present in MDA-MB-231 and MCF-7 cell extracts using the results of SQT-PCRs on both cell batches (except for scn9a, amplified from only one of the two MCF-7 cell line batches, and scn5A, which was apparently expressed too lowly in one MCF-7 batch for CNe to be calculated). Assuming that the PCR reactions performed on strongly and weakly metastatic cell RNA extracts had similar efficiencies, differences in the calculated CNt and CNe values would reflect real differences in expression levels.
NADH-cytochrome b5 reductase (Cytb5R), which is expressed at very similar levels in normal, cancerous and strongly metastatic cells derived from numerous tissue types (20; Fitzsimmons, S. A., et al (1996) J. Natl. Cancer Inst. 88, 259-269; Marin, A., et al (1997) Br. J. Cancer 76, 923-929), was present in both rat and human degenerate primer screens as a major constituent of the non-specific products found (the “non-VGSCα” clones). Consequently, this was used as a control amplicon in SQT-PCRs, ie to control for the effects of variations in quality and quantity of the initial RNA, efficiency of the reverse-transcription and amplification between samples (primers are shown in Table 1B). Cytb5R 20-mer primers amplified nucleotides 385-809 and 299-790 of rat and human homologues, respectively (annealing temperature, 60° C. for both).
PCR tests on breast biopsy tissue. 0.1-0.5 g pieces of frozen tissue were chopped into small pieces using a sterile scalpel and forceps and placed in a cold, glass homogenizer. Total cellular RNA was then isolated as described above. RNA quality was preliminarily assessed by gel electrophoresis and quantity determined by spectrophotometric analysis.
RNA extracts were then used as the template for sscDNA synthesis, performed as above. The possible expression of scn5A, scn8a and scn9a RNAs in the biopsy samples was tested by PCR, using the same primers as for the specific PCRs (Table 1B). hCytb5R specific PCR tests were also carried out to further control for the quality of the extracted RNA; samples which did not yield evident hCytb5R products were rejected. PCRs were performed, using 2.5 μl of the synthesised sscDNA, 0.2 millimolar dNTPs, 1 micomolar of each specific primer and 1 unit of Taq, under the following conditions: 94° C. for 5 min; 1 U enzyme added; 94° C. for 1 min; 59-62° C. for 1 min (depending on the primer pair); 72° C. 1 min; final incubation at 72° C. for 10 min with the main section repeated 30-60 times (depending on the primer pair). PCR reactions with no template added were also performed to control for cross-contamination from other DNA sources. 5 μl aliquots of the final reaction were analysed by gel electrophoresis on 0.8% agarose gels.
PCR tests were carried out on each of at least two cDNA templates (except for sample 1, from which only 5 μg of RNA was obtained), manufactured independently from the same RNA extract, thus controlling for possible variability in cDNA manufacture and PCR efficiency.
Data analysis. All quantitative data were determined to be normally distributed and are presented in the text as means±standard errors. Statistical significance was determined with Student's t test or χ2 test, as appropriate.
GenBank sequence nucleotide numbers. Nucleotide numbering was according to accession numbers M77235, AB027567, X82835, Y09501 for scn5a, scn8a, scn9a and hCytb5R, respectively.
Results
Electrophysiological studies. The average resting potential of MDA-MB-231 cells was −18.9±2.1 mV (n=27; range −12 to −61 mV) which was significantly more depolarised than the value of −38.9±2.5 mV (n=26; range −8 to −51 mV) for the MCF-7 cells (p<0.001). The membrane capacitance of the MDA-MB-231 cells was 28.5±2.7 pF (n=35; range 14.7 to 76.6 pF) which was significantly smaller than the value of 36.9±2.8 pF (n=38; range 13.5 to 90.0 pF) for the MCF-7 cells (p<0.05).
29% of the MDA-MB-231 cells tested (n=16/56) expressed an inward current of up to 600 pA in amplitude (FIG. 1A), which corresponded to a current density of 5.6±0.5 pA/pF (n=16). The inward currents activated at 41.3±2.4 mV (n=4), peaked at −6.3 2.4 mV (n=4; FIG. 1C) and were abolished in Na+-free medium (not shown; n=2), consistent with them being VGS currents. In contrast, none of the MCF-7 cells tested (n=72) showed an inward current (FIG. 1B).
The VGS current was suppressed partially by micromolar TTX (FIG. 2A). The effect of the toxin was concentration dependent in the range 100 nM-6 μM (FIG. 2B). However, even at the highest concentration used (6 μM), only 64.7±6.1% of the current was blocked by TTX (n=5). There was a small (9±3%) reduction in peak current with 100 nM TTX, which was significant (p<0.05), indicating that a minor, TTX-sensitive (TTX-S) component was also present (FIG. 2B).
Voltage-gated outward currents were also recorded. 100% of the MCF-7 cells tested (n=72) expressed large outward currents of up to 7 nA in amplitude (FIG. 1B), which corresponded to a current density of 27.4±4.9 pA/pF (n=33). These outward currents activated at −9.2±1.9 mV (n=12) and showed a peak amplitude of 1081.1±264.7 pA at +90 mV (n=12). The current was reduced to 34.3+5.4 pA (n=15; p<0.01), i.e. by 97%, by substituting Cs+ for K+ in the internal pipette solution. In comparison, MDA-MB-231 cells showed much smaller outward currents of up to 150 pA (n=35; FIG. 1B), which corresponded to a current density of only 2.6±0.4 pA/pF (n=13; p<0.01 cf. comparable currents recorded in the MCF-7 cells).
VGSCα mRNA expression in the cell lines. The results of the degenerate-primer screens for the different cell line RNA extracts are shown in Table 2. Two VGSCαs were identified in the screens on the strongly metastatic cell line: products of SCN5A and SCN9A VGSCα genes. In contrast, scn8a was the only VGSCα found in the degenerate screens of the weakly metastatic MCF-7 cells. It has previously been shown that for Nav1 VGSCαs, the proportion of clones in degenerate primer screens representing each VGSCα type reflects the actual proportion of that subunit within the cellular VGSCα mRNA pool (21). Thus, in the strongly metastatic cells, screen results indicated that scn5a (56.0±4.0%) was expressed at a much greater level than scn9a (12.0±4.0%) and scn8a (0%) (Table 2).
TABLE 2Summary of the VGCSα degenerate primer screen results.Results are shown as percentage of clones tested (n = 25in each case). Each screen is the result of two extracts fromeach cell line. Errors indicate standard errors.VGSCαMDA-MB-231MCF-7Scn5a56.0 ± 4.00Scn8a0 2.0 ± 2.0Scn9a12.0 ± 4.00Non - VGSCα32.0 ± 0  98.0 ± 2.0
Primer-specific PCRs yielded products for scn5, scn8a and scn9a (as well as for hCytb5R) in both cell lines, indicating that all of these mRNAs were expressed in both MDA-MB-231 and MCF-7 cells. However, scn5a and scn9a required markedly less amplification (CNt) to yield detectable products and reach CNe in SQT-PCRs on MDA-MB-231 vs. MCF-7 cell extracts, indicating an overall greater level of expression in the strongly metastatic cells (FIGS. 3A and 3C). Importantly, the most striking, consistent difference was seen for SCN5A: CNt=24.75±0.48 vs. 37.50±1.56; CNe=28.36±0.46 vs. 38.54±0.14, for MDA-MB-231s vs. MCF-7 cells, respectively (FIG. 3A). Assuming an 80% PCR efficiency (21), this would indicate −1800-fold difference in expression levels between the two cell lines.
Scn9a was more readily amplified in the strongly metastatic (CNt=30.75±0.63; CNe=34.44±0.65) than the weakly metastatic cells (CNt=42.5±4.5; CNe=46.0±3.2), but this TTX-S VGSCα was not as prominent as scn5a in degenerate screens, indicating a lower level of expression. In contrast, hCytb5R ‘control’ and scn8a SQT-PCRs showed very similar levels of expression in both MDA-MB-231 and MCF-7 cells (FIGS. 3B and 3D): CNt=20.25±0.25 vs. 22.0±0.56, CNe=23.96±1.00 vs. 25.16±0.34, for hCytb5R; CNt=33.25±0.25 vs. 32.75±0.63, CNe=36.85±0.32 vs. 35.61±0.49, for scn8a. Importantly, hCytb5R was the major constituent of the ‘non-VGSCα’ clones found in the degenerate screens, representing almost all of the non-VGSCα clones (equivalent to 28.0±0% of all clones) in the MDA-MB-231 cells and 54.0±6.0% of all the clones in the MCF-7 cell line screen. The increased incidence of this non-VGSCα clone in the degenerate screens of the MCF-7 cells is consistent with a lower VGSCα target to noise ratio in these cells compared to their strongly metastatic counterpart, also evident from the SQT-PCR data.
The MDA-MB-231/MCF-7 VGSCα sequences obtained have been submitted to GenBank.
VGSCα mRNA expression in breast biopsy tissue. RNA was extracted successfully, with positive hCytb5R tests obtained, from 12 samples. Generally, PCR results of the VGSCα and hCytb5R control tests were readily repeatable across different synthesised cDNA batches. The results obtained are summarised in Table 3. All three VGSCα genes found to be expressed in the cell lines were detected in the biopsy samples, confirming the conservation in vivo of the VGSCα expression profile of the in vitro models. Several SCN8A and SCN9A products (corresponding to different splice forms of these genes; (21)) were amplified from all samples (FIGS. 4D and E), as was the hCytb5R control (FIG. 4F), except in sample 6. It is likely that the RNA extracted from this sample was significantly more degraded than the other samples, as evidenced by the greater number of PCR cycles required to amplify the hCytb5R control product (40 not 30 cycles). There was, however, no evident correlation between scn8a or scn9a expression and lymph node metastasis (LNM). In contrast, expression of scn5a was strictly sample-dependent (FIGS. 4A and B). All evident products of these tests were cloned and sequenced, and it was verified that these products were truly derived from SCN5A. Scn5a is VGSCα sequences obtained from these samples have been submitted to GenBank.
Accession numbers of submitted sequences are as follows:    Accession#: AJ310882    Description: Homo sapiens partial mRNA for voltage-gated sodium channel    Nav1.7 (SCN9A gene) cell line MDA-MB-231    Accession#: AJ310883    Description: Homo sapiens partial mRNA for Nav1.7 voltage-gated-sodium    channel (SCN9A gene) cell line MCF-7    Accession#: AJ310884    Description: Homo sapiens mRNA for Nav1.6 voltage-gated-sodium (SCN8A gene) Nav1.6, D3 neonatal splice variant, cell lines MDA-MB-231    Accession#: AJ310885    Description: Homo sapiens partial mRNA for voltage-gated sodium channel    Nav1.6 (SCN8A gene), D3 neonatal splice variant, cell line MCF-1    Accession#: AJ310886    Description: Homo sapiens partial mRNA for voltage gated sodium channel    Nav1.5 (SCN5A gene), D1 neonatal splice variant, cell line MDA-MB-231    Accession#: AJ310887    Description: Homo sapiens partial mRNA for voltage-gated sodium channel    Nav1.5 (SCN5A gene) D1 neonatal splice variant, cell line MCF-7    Accession#: AJ310888    Description: Homo sapiens partial mRNA for voltage gated sodium channel    Nav1.5 (SCN5A gene), D1 neonatal splice variant, biopsy sample 2    Accession#: AJ310889    Description: Homo sapiens partial mRNA for voltage-gated sodium channel    Nav1.5 (SCN5A gene), D1 neonatal splice variant, biopsy sample 3    Accession#: AJ310890    Description: Homo sapiens partial mRNA for voltage-gated sodium channel    Nav1.5 (SCN5A gene) D1 adult splice variant, biopsy sample 1    Accession#: AJ310891    Description: Homo sapiens partial mRNA for voltage gated sodium channel    Nav1.5 (SCN5A gene), D1 adult splice variant, biopsy sample 7    Accession#: AJ310892    Description: Homo sapiens partial mRNA for voltage-gated sodium channel    Nav1.5 (SCN5A gene), biopsy sample 6    Accession#: AJ310893    Description: Homo sapiens partial mRNA for voltage gated sodium channel    Nav1.5 (SCN5A gene), D1:S3 exon-skipped splice variant, biopsy sample 8    Accession#: AJ310894    Description: Homo sapiens partial mRNA for voltage-gated sodium channel    Nav1.5 (SCN5A gene), D1 neonatal splice variant, biopsy sample 4    Accession#: AJ310895    Description: Homo sapiens partial mRNA for Nav1.5 (scn5a/h1) voltage-gated    sodium channel (SCN5A gene), D1 neonatal splice variant, biopsy sample 5    Accession#: AJ310896    Description: Homo sapiens partial mRNA for voltage-gated sodium Nav1.5    (SCN5A gene) (SCN5A gene), cell line MDA-MB-231    Accession#: AJ310897    Description: Homo sapiens partial mRNA for voltage-gated sodium Nav1.7    (SCN9A gene)(SCN9A gene), cell line MDA-MB-231    Accession#: AJ310898    Description: Homo sapiens partial mRNA for voltage-gated sodium channel    Nav1.6 (SCN8A gene), cell line MCF-7    Accession#: AJ310899    Description: Homo sapiens partial mRNA for NADH-cytochrome b5 reductase    (B5R gene) cell line MDA-MB-231    Accession#: AJ310900    Description: Homo sapiens partial mRNA for NADH-cytochrome b5 reductase    (B5R gene) cell line MCF-7
Sequences have also been submitted for the following:    scn5a MDA-MB-231 SQT-PCR sequence    scn5a MCF-7 SQT-PCR sequence    scn9a MDA-MB-231 SQT-PCR sequence (3UTR)    scn9a MCF-7 SQT-PCR sequence (3UTR)
Some specific points regarding the SEQUENCE data:    SCN5A—Three nucleotide differences from the published sequence (GenBank M77235) in the sequence obtained outside the D1 neonatal exon: 689/690 (CT to GC) differences would substitute an alanine for a glycine residue at amino acid position 180 (all numbering according to M77235). All other voltage-gated sodium channel alpha subunit genes have a glycine at this residue, and thus it is most probable that the published sequence (M77235) contains sequence errors at this location. 992 (T to C) difference would not change the amino acid sequence and may represent a natural, silent-polymorphism in the SCN5A gene.    SCN8A—No nucleotide differences from our previously published sequences [SCN8A in prostate cancer cell lines] (GenBank AJ276141 and AJ276142).    SCN9A—No nucleotide differences from the published sequence (GenBank X82835).    The SCN5A D1 neonatal exon—this could be clinically important. This is the first report of the existence of an apparent alternative splice form of scna5a at this location. The SCN5A gene structure has been investigated previously (Wang et al., 1996), using scn5a cDNA sequences to probe a human genomic library but alternative exons for D1S3 were not found, presumably because the hybridizing cDNAs were of the known adult rather than the neonatal form. The scn5a neonatal form differs from the previously published adult form (Gellens et al., 1992; GenBank M77235) at 31 of the 92 nucleotides in this conserved exon. These 31 nucleotide differences in the neonatal SCN5A form result in 7 amino acid substitutions, many more than observed for the other VGSC alpha subunit genes studied thus far.
To date, alternative splicing of neonatal and adult exons has been found in D1S3 in four other VGSC alpha genes: SCN2A, SCN3A, SCN8A and SCN9A. In each of these instances the alternative exons have 19-21 nucleotide differences, which result in 1-2 amino acid substitutions. One amino acid substitution at residue seven of this exon is consistent across all of these genes: the substitution of an aspartate residue in the adult form to a neutral amino acid in the neonatal form. Alternative splicing in scn5a was not completely consistent with D1S3 splicing previously described for other VGSC alphas in two main ways:    (1) In the scn5a neonatal form, the aspartate residue in the adult form was not substituted for a non-charged amino acid, but a positively charged lysine residue.    (2) The 31 nucleotide differences in the neonatal scn5a result in 7 amino acid substitutions, many more than the 1-2 amino acid substitutions observed for the other VGSC alpha genes with alternative splicing at D1S3, previously studied.
TABLE 3Summary of results of specific RT-PCR tests on breast cancerbiopsy samples. (+) indicates that a specific productwas obtained; (−) indicates that no specific product wasamplified; NT that the test was not performed; ND that thegrade of the tumour was not determined. PCR tests wereperformed for up to 55, 50 and 50 cycles for scn5a, scn8a andscn9a, respectively. hCytb5R tests were performed for 30 cyclesexcept for sample 6, for which 40 cycles were used (denotedby *). Clinically assessment lymph node metastasis (LNM)and tumour grade are also shown for each case. ForLNM (+), the values in parentheses indicate the numberof lymph nodes which were determined as positive/numberof nodes examined.SampleClinical GradehCytb5Rscn8a scn9aScn5aLNM12+NT+++(4/4)23+++++(3/7)32+++++(3/7)42+++++(8/12)5ND+++++(8/13)62+(*)−−++(9/14)71++++−(0/22)82++++−(0/13)92+++−−(0/15)103+++−−(0/10)111+++−−(0/15)123+++−−(0/9)
A ‘double-blind’ test associating scn5a expression with LNM revealed that these two characteristics were directly correlated in 10 out of the 12 (83%) cases examined, giving combinations of scn5a+/LNM+ (n=6) and scn5a−/LNM− (n=4) (χ2=6.0; df=1; 0.02>p>0.01; Table 3). One of the two exceptions where the sample was scn5a+but apparently LNM− (sample 7) was interesting in that the patient subsequently relapsed, developing distant metastases within three years of the preliminary diagnosis. Whether relapse also occurred for the patient who provided the other exceptional case (sample 8) could not be determined.
Discussion
The present study shows (i) that strongly, but not weakly metastatic breast cancer cells displayed VGS currents, almost entirely composed of a TTX-resistant (ITX-R) component; (ii) that a particular TTX-R VGSCα gene, SCN5A, was predominantly expressed in strongly metastatic cells, but expressed at only very low levels in weakly metastatic cells; and (iii) that scn5a expression in biopsy samples correlated strongly with clinically assessed lymph node metastasis. Furthermore, the high-level VGSC expression was accompanied inversely by much reduced outward currents in the cell lines, and a relatively depolarised resting potential. Taken is together, these characteristics would render metastatic cell membranes potentially ‘excitable’, consistent with their hyperactive behaviour.
Scn5a expression is associated with breast cancer metastasis. The electrophysiological and RT-PCR results demonstrated consistently that SCN5A gene products (also termed h1 or SkM2) were predominantly expressed in the strongly metastatic cell line (FIG. 5) and were associated with breast cancer metastasis in vivo. We have shown previously for human and/or rat prostate cancer cells that VGSC activity contributes to cellular behaviours integral to metastasis, including cellular process extension (7), lateral migration (8), transverse invasion (5,6,9) and secretory membrane activity (10). Subsequently, scn9a was identified as the ‘culprit’ VGSCα (21). In the present study, the correlation of scn5a expression with increased cellular metastatic potential in vitro, and lymph node metastasis in vivo, would strongly indicate a significant role for scn5a activity in the metastatic behaviour of breast cancer cells.
Although the present study is the first to associate scn5a with cellular metastatic potential, others have previously reported expression of this VGSCα in cancer cell lines. Scn5a mRNA and functional protein expression have been shown to occur in B104 neuroblastoma cells (25) and RT4 peripheral neurotumour cancer cell lines (26,27). At present, it is not clear why strongly metastatic cells from carcinomas derived from different tissues should specifically upregulate the expression of different VGSCαs. Also, it is not known if, amongst the various VGSCαs, only scn5a would be capable of potentiating metastasis in breast carcinoma. If so, then it may be that this ability results from characteristics peculiar only to this VGSCα. Such possible, characteristic features of scn5a include the following: (i) Possession of C-terminal PDZ domains (28,29), potentially enabling particular interaction with the cytoskeleton; (ii) extremely low level of protein glycosylation (5% of the total protein mass, compared to up to 40% protein mass in other VGSCαs; (30)); (iii) highly promiscuous ion selectivity in given conditions, allowing Ca2+ entry (31); (iv) very slow activation and inactivation kinetics (28,32); and (v) regulation of expression by steroid hormones (33).
Another notable characteristic of scn5a is that its expression appears to be under very tight spatio-temporal control and highly dynamic regulation. SCN5A gene products are classically expressed at very high levels in cardiac and neonatal/denervated skeletal muscle (29,34). However, Scn5a mRNA has also been detected in non-excitable, cultured spinal cord astrocytes (35) but not in a variety of cell types which express almost all other VGSCαs, like dorsal root ganglion neurones (26,36,37). Furthermore, in skeletal muscle particularly, significant changes in expression levels can occur over a period of only days after birth (34) and in response to denervation (38).
Conservation of breast cancer VGSCα expression in biopsy tissue. The profile of VGSCα expression in weakly and strongly metastatic breast cancer cells that we have obtained from the two cell lines (FIG. 5) is consistent with the results of the PCRs performed on the biopsy tissues. All three VGSCαs found in the cell lines were found to be expressed in the tissue samples and the expression of the predominant scn5a type was correlated strongly with the surgically characterised metastases.
Although relative expression levels of scn5a, scn8a and scn9a cannot directly be determined from the PCR tests, the apparent ease of amplification of the different VGSCαs from the biopsy tissues is consistent with the biopsy samples consisting almost entirely of a mass of essentially non-metastatic primary tumour cells with only a very small number of strongly metastatic cancer cells present in malignant tumours (e.g. 39,40). Thus, scn8a and scn9a (which are expressed at greater levels than Scn5a in weakly metastatic cells) could be detected in biopsy tissue using a lower number of PCR cycles, compared with scn5a, even from samples displaying evident lymph node metastasis.
The PCRs performed on the biopsy tissues did not yield reliable quantitative information concerning expression levels of the various VGSCαs, mainly due to the large variability in the quality of extracted RNA from one sample to another, as monitored by the control RNA. Scn5a expression was apparently so low in weakly metastatic cells that it could not be detected in non-malignant biopsy tissues. However, the expression, being greatly upregulated in the strongly metastatic cells within the biopsies, became readily detectable by PCR.
Multiplicity of VGSCα expression in breast cancer cell lines. The expression of multiple VGSCα genes was determined in both breast cancer cell lines and is consistent with the relative VGSCα expression profiles illustrated in FIG. 5. In brief, the level of scn8a was similar for both cell lines but very low, whilst expression of scn5a and scn9a were significantly greater for the MDA-MB-231 cell line. In particular, scn5a expression accounted for >80% of the VGSCαs in these cells. A D1 neonatal splice form of SCN5A may be of clinical importance, as discussed above. Multiplicity of VGSCα expression has also been found in rat and human prostate cancer cell lines of differing metastatic potential (21). The pharmacological data (TTX blockage) indicated that the VGS currents detected in the MDA-MB-231 cells were mainly TTX-R (IC50>1 μM). This is consistent with the determined mRNA expression profile of these cells in which the TTX-R scn5a VGSCα is the predominant channel. The scn9a VGSCα expressed, but at much lower levels (FIG. 5), would yield TTX-S currents which could contribute to the TTX sensitivity observed at lower (100 nM) concentrations. Possible consequences of multiple VGSCα expression have been discussed previously (21). Interestingly, the full-length scn8a products detected in both strongly and weakly metastatic breast cancer cells were the neonatal splice form as determined by the product size (Diss, J. K. J., unpublished observation). This form of scn8a codes for a highly truncated VGSCα protein, and has been found to be preferentially expressed in neonatal and non-excitable adult tissues (41). Neonatal scn8a is thought not to be capable of creating functional VGSCs, instead acting as a “fail-safe” mechanism, preventing the functional expression of leakily expressed, non-truncated scn8a VGSCαs. The detection of neonatal scn8a mRNA in biopsy samples (as determined by the product size; FIG. 4D) indicates that this mechanism is also present in vivo.
VGSC expression in breast and prostate cancer: Comparative aspects. Many aspects of the findings of this study are similar to those determined using similar techniques in rat and human prostate cancer cell lines of differing metastatic potential (5,6,21): (i) the strongly metastatic cells had relatively depolarised resting potentials (6). (ii) VGS currents were detected in a sub-population of strongly metastatic cells (54% MAT-LyLu, 10% PC-3, 29% MDA-MB-231) and never detected in corresponding weakly metastatic cells (AT-2, LNCaP, MCF-7); (iii) VGSCα mRNA was detected in cells of both strong and weak metastatic potential, but with greater expression in strongly metastatic cells; (iv) multiple VGSCα expression was determined in all cells; (v) all cells expressed scn8a, mainly in the non-functional, neonatal form (21); and (vi) the predominant VGSCα (scn9a in prostate cancer cells; scn5a in breast cancer cells) was expressed more than 1000-fold more in strongly vs. weakly metastatic cells.
That we should find a similar mechanism potentially involved in metastasis of both breast and prostate cancer is not completely surprising in view of their similarities in tumour biology (e.g. hormone-responsiveness and propensity for bone metastasis) but does strongly encourage future work to investigate VGSC activity and metastasis in other cancer types. VGSCα expression has been determined in developing small cell carcinoma of the lung (42) and gliomas (43,44). Thus, functional VGSC expression may be part of a general mechanism for cancer progression and metastasis.
On the other hand, it is unclear why a specific, but different VGSCα should be associated with metastasis in breast and prostate cancers. Whilst not intending to be bound by theory, it is possible that all VGSCαs may have the capability of potentiating the metastatic cascade, or only specific types (including scn5a and scn9a). All VGSCαs that are capable of potentiating metastasis may affect the same basic cellular process(es) within the cascade, for example cellular process extension, lateral migration, secretion or transverse invasion. The specific association of a particular VGSCα with metastasis in a given cancer type may result from tissue- (or cancer-) specific transcriptional regulation/control mechanisms, for example androgens in prostate cancer or oestrogen in breast cancer. Alternatively, this specific association may result from different VGSCα(s) affecting different cellular processes which may be more or less important for successful metastasis from different primary tumour sites.
Clinical implications. Prior to the present invention, only indirect indicators as to the likelihood of metastatic potential were available, since, although it is possible to detect micrometastases in a proportion of patients with breast cancer, many patients who do not have micrometastases at presentation eventually develop overt metastatic disease during follow-up (45). Consequently, clinicians, therefore, require a more accurate method for predicting the likelihood of development of metastatic disease, and the presence of VGSCs could act as an independent prognostic parameter in a multivariant approach to this problem. Of perhaps greater significance in the future is the potential implications of inhibiting VGSC activity. The scn5a VGSCα is already the specific target of numerous anti-arrhythmic and anti-convulsant drugs, since dysfunction of scn5a in cardiac tissue is intricately linked to several forms of heart disease and arrhythmia (46). Interestingly, the breast cancer drug tamoxifen has been found also to protect the heart (47) although it is not known if this involves VGSC modulation (48). The present work, which identifies scn5a as the potential ‘culprit’ VGSCα in breast cancer metastasis, therefore, indicates that scn5a-specific drugs may be inhibitors of the metastatic cascade.
Sequence information The question of whether there is any difference in the sequences of the ‘wild type’ and the ‘breast cancer culprit’ SCN5A gene is an important one. In general, there are two major reasons that suggest that differences in sequence are less important than differences in level of expression: (a) The sequence data that we have obtained so far shows identity (note however that our data represent at the most only some 17% and often less than 10% of the whole sequence) and (b) the expression levels are >1000-fold different between the strongly vs the weakly metastatic cells. Taken together, we think that it is the level of expression (and whatever is responsible for it) rather than sequence difference(s) that is important. Of course, there may be some sequence differences that are important for cancer. To test that would require complete sequencing of the gene which is not a trivial exercise. There are examples of quite subtle nucleotide changes in VGSC genes giving rise to profound changes in function, leading to a pathological condition [see J. Physiol. (2000) 529:533-539, for a recent example].